Variations in the breeding behavior of cichlids and the evolution of the multi-functional seminal plasma protein, seminal plasma glycoprotein 120.

BACKGROUND: Seminal plasma proteins are associated with successful fertilization. However, their evolutionary correlation with fertilization mechanisms remains unclear. Cichlids from Lake Tanganyika show a variety-rich spawning behavior that is associated with the transfer of the sperm to the egg for fertilization. One of these behaviors, called "oral fertilization," emerged during their speciation. In oral fertilization, females nuzzle the milt from male genitalia and pick up the released eggs in their mouths, which are then fertilized inside the oral cavity. Thus, the success of the fertilization is dependent on the retention of sperm in the oral cavity during spawning. Sperm aggregation and immobilization in viscous seminal plasma may help retain the sperm inside the oral cavity, which ultimately determines the success of the fertilization. Seminal plasma glycoprotein 120 (SPP120) is one of the major seminal plasma proteins present in cichlids. SPP120 has been implicated to immobilize sperm and increase the milt viscosity. However, the functional linkage between oral fertilization and seminal plasma proteins has not been investigated. RESULTS: During trials of simulated oral fertilization, it was observed that milt viscosity contributed to fertilization success by facilitating longer retention of the milt inside the mouth during spawning. Glycosylation of SPP120 was associated with high milt viscosity. Its glycosylation was specifically present in the milt of cichlid species exhibiting oral fertilization. Moreover, recombinant SPP120 from several the oral fertilization species strongly immobilized/aggregated sperm. Therefore, the functions of SPP120 (immobilization/aggregation and its glycosylation) may contribute to success of oral fertilization, and these functions of SPP120 are more prominent in oral fertilization species. In addition, comparative phylogenetic analyses showed a positive evolutionary correlation between SPP120 function and oral fertilization. Hence, these evolutions may have occurred to keep up with the transition in the mode of fertilization. In addition, rapid evolution in the molecular sequence might be associated with functional modifications of SPP120. CONCLUSION: These results suggest that SPP120 might be associated with oral fertilization. In other words, reproductive traits that define the interaction between sperms and eggs could be the evolutionary selective force that cause the rapid functional modification of the fertilization-related reproductive protein, SPP120.

A Unique Seminal Plasma Protein, Zona Pellucida 3-Like Protein, has Ca2+ -Dependent Sperm Agglutination Activity.

Identification of seminal proteins provides a means of investigating their roles. Despite their importance in the study of protein function, such as regulation of sperm motility, it is difficult to select candidates from the large number of proteins. Analyzing the rate of molecular evolution is a useful strategy for selecting candidates, and expressing the protein allows the examination of its function. In the present study, we investigated seminal plasma proteins of the cichlid Oreochromis mossambicus, which exhibits a unique mode of fertilization and a rapidly evolving gene that encodes a seminal plasma protein, zona-pellucida 3-like (ZP3-like), which does not belong to the same molecular family as other ZPs. Seminal plasma proteins of O. mossambicus were separated by two-dimensional electrophoresis, and 19 major proteins were identified by mass spectrometry (MALDI-Tof Mass). Because proteins that are under positive selection often impact sperm function, the rates of molecular evolution of these proteins were analyzed in terms of non-synonymous/synonymous substitutions (ω). Among the 19 proteins, positive selection was supported for five genes; functional assays were carried out on four of the proteins encoded by these genes. Of the four positively selected proteins, only ZP3-like protein agglutinated sperm in a dose- and Ca2+ -dependent manner. The other three proteins did not affect sperm motility. Because of the unique fertilization type, in which fertilization occurs in the buccal cavity, the need to retain sperm within the cavity during spawning, and the agglutination of sperm, which may be partly assisted by ZP3-like protein, may contribute to fertilization success. Fertilization in the buccal cavity may be related to its rapid molecular evolution.